The present invention relates to a novel uricase and a method for the preparation thereof. Uricase is an enzyme useful as a reagent in the quantitative determination of uric acid and the present invention provides a novel uricase having properties quite suitable for this purpose, which can be produced by a thermophilic microorganism.
Uricase (EC 1.7.3.3) is an enzyme found in the livers and kidneys of animals as well as certain microorganisms and catalyses the oxidation of uric acid in the presence of oxygen so that it is used widely in the quantitative determination of uric acid in clinical examinations.
As is disclosed in Japanese Patent Kokai Koho Nos. 55-81586 and 56-124381, Japanese Patent Publication No. 56-43230, uricase is widely produced by a microbiological means while one of the problems in these microbiological processes is that the culturing of the microorganism takes a considerably long time of one day to three days or even longer because the microorganism utilized in the process disclosed in each of the above mentioned patent literatures is mesophilic.
While it would be an advantageous way to utilize a thermophilic microorganism exhibiting a high rate of metabolism and capable of rapid growth in respect of the possibility of decreasing the time taken for the microbiological production of uricase, no such a process has yet been reported in which a thermophilic microorganism is utilized for the production of uricase. It would also be an advantageous way to utilize a thermophilic microorganism as a source of uricase in respect of the excellent stability of the produced enzyme.
As viewed from the standpoint of the activity characteristics of a uricase, it is desirable that the uricase should have a high activity in the pH range including or in the vicinity of pH 6.5 in order to reduce the amount of the enzyme required in the clinical analysis for uric acid because the common determination of uric acid is performed by the coupled reaction with peroxidase from horseradish which exhibits the optimum activity at a pH of about 6.5. Nevertheless, the uricases conventionally used in the prior art have an optimum pH in the range of 8.5 to 9.0 and can exhibit substantially no activity at a pH of 6.5 (see Japanese Patent Publication No. 56-43230) or the activity at a pH of 6.5 is lower than 40% of the activity at a pH of 8.5 to 9.0 [see Agric. Biol. Chem., volume 44(12), page 2811 (1980)].
In view of the above described problems in the prior art in connection with uricases, the inventors have conducted extensive searching works to discover a thermophilic microorganism capable of producing a uricase having activity in a wide pH range among the thermophilic microorganisms separated from a large number of soil samples and finally reached a discovery leading to the establishment of the present invention.